No abstract

Purpose BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. Experimental Design The constitutively active BRAFV600E allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16INK4a levels in pilocytic astrocytoma. Results BRAFV600E expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAFV600E-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16INK4a whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16INK4a had significantly shorter overall survival. Conclusions BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16INK4a expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.

SUMMARY Ubiquitin-specific proteases (USPs) constitute the largest family of deubiquitinating enzymes, whose catalytic competency is often modulated by their binding partners through unknown mechanisms. Here we report a series of crystallographic and biochemical analyses of an evolutionarily conserved deubiquitinase, USP12, which is activated by two β-propeller proteins, UAF1 and WDR20. Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far away from its catalytic center. Without increasing substrate affinity of USP12, the two β-propeller proteins potentiate the enzyme through different allosteric mechanisms. UAF1 docks at the distal end of the USP12 Fingers domain and induces a cascade of structural changes that reach to a critical ubiquitin-contacting loop adjacent to the catalytic cleft. By contrast, WDR20 anchors at the base of this loop and remotely modulates the catalytic center of the enzyme. Our results provide a mechanistic example for allosteric activation of USPs by their regulatory partners.